Design, Synthesis and Molecular Modeling Studies of Thiosemicarbazone & Thiazole Derivatives as Potential Anti-Malarial Agents

A series of novel thiosemicarbazone & thiazole derivatives (Kp1-10) have been designed, synthesized and evaluated for potential anti-malarial activity. The antimalarial activity of the synthesized thiazole derivatives (Kp1-10) was assessed against human pathogenic malarial strain viz. Plasmodium falciparum while quinine was taken as the standard drug. compound Kp-9 was found to be most promising which exhibited strongest inhibitory activity against P. falciparumwith an IC 50 value of 0.29µg/mL which was higher than the reference drug quinine (1.26µg/mL). The SAR studyrevealed that thesubstitution with electron withdrawing group at phenyl increases anti-malarial activity as shown in compound Kp-9. The result of molecular docking studies showed that compounds Kp-9, Kp-1, Kp-3, Kp-4 showed good docking scores with protein (PDB code: 5TBO). The compound Kp-9 showed highest docking score (-9.519). Whereas, compounds Kp-1, Kp-3, Kp-4 and Kp-10 showed good docking scores (-8.764, -8.406, -9.062, -9.435 respectively) with critical interactions with the amino acid residues such as VAL532, ILE237, LEU531, HIE185, TYR528, ASN274, ARG265. The results of biological activity and docking study revealed that the presence of electron withdrawing group at 4th position of phenyl ring attached is crucial for better anti-malarial activity and favorable drug-like prole which can emerge as a potential drug molecule in further development.


Introduction
Protozoan parasite existence was discovered even in millions of years old fossil sponges. Protozoan parasites are harmful to humans and cause one million deaths yearly [1]. While there is little doubt that the protozoan disease burden is focused in tropical and subtropical regions of the world, more temperate areas of our globe, including North America and the Asia Paci c region, are also in uence by protozoan diseases such as Malaria, Leishmaniasis, Filariasis, Cryptosporidiosis, African trypanosomiasis, Chagas disease [2]. Here, we only focused on malaria which is a life threatening disease. According to World health organization (WHO) 219 million cases of malaria and 435,000 related deaths occurred in 2017 in the world malaria report 2018. India apparently accounts for 4% of the worldwide malaria burden and represents 87% of Southeast Asia's overall malaria cases [3,4]. Six countries accounted for more than half of all malaria cases worldwide: Nigeria (25%), the Democratic Republic of the Congo (12%), Uganda (5%), and Côte d'Ivoire, Mozambique and Niger (4% each) [5]. Malaria is a vector-borne, tropical parasite disease occurring worldwide in 91 countries. The condition is triggered by the protozoal parasites of the genus Plasmodium [6,7]. The most common species are Plasmodium falciparum and Plasmodium vivax, for which humans are the only mammalian hosts, and they are mostly responsible for the most signi cant public health burden [8,9]. In addition to the few therapeutic options available, effective treatment of a protozoal disease is an enormous problem due to adverse effects, medication toxicity, resistance, long-term therapies, the susceptibilities of individual systems, and the parasite variability [10,11].
Thiazole and thiosemicarbazone derivatives are widely described in the literature and present a wide range of biological activities [12]. There are various reasons that make thiazole scaffolds interesting prototypes are their accessible chemical synthesis, low costs pertaining to reactivity, good yield reactions, and ability to generate a series of analogues, among others [13]. Thiazole motif has been widely explored by various researchers in the eld of medicinal chemistry to combat various illnesses. In the normal functioning of the nervous systems, thiazole containing vitamin B1 (thiamine), for instance, helps to synthesize acetylcholine [14,15]

General procedure for the synthesis of thiazole derivatives (2) (Kp1-10)
To a solution of 1 equivalent of thiosemicarbazone in isopropyl alcohol was added in 1 equivalent of phenacyl bromide. The resulting mixture was kept under re ux for 5 hours. After cooling at room temperature, the formed precipitates was ltered and washed with saturated solution of NaHCO 3 followed by cold distilled water. Final product was recrystallized in ethanol. the purity of the product as well as composition of reaction mixture were monitored by TLC (Thin layer chromatography) using solvent n-hexane:ethyl acetate(3:2).
Light brown precipitates; Yield 72%; mp. 210-212°C; Rf 0.6 was also determined IR (KBr) Vmax(cm − 1 ): 620 cm − 1 (C-S), 1168 cm − 1 (C-N), 1606 cm − 1 (C = C), 3050 cm − 1 (C-H Aromatic), 3127 cm − 1 (N-H), 3416 The synthesized thiazole derivatives (Kp1-10) were evaluated for their anti-malarial activity and their results was expressed in terms of IC 50 µg/ml and are presented in Table1. The in vitro anti-malarial assay was carried out in 96 well microtitre plates according to the micro assay protocol of Rieckmann and coworkers with minor modi cations. The cultures of Plasmodium falciparum strain were maintained in RPMI-1640 medium supplemented with 25mM HEPES, 1% D-glucose, 0.23% sodium bicarbonate and 10% heat inactivated human serum. The asynchronous parasites of Plasmodium falciparum were synchronized after 5% D-sorbitol treatment to obtain only the ring stage parasitized cells. For carrying out the assay, an initial ring stage parasitaemia of 0.8 to 1.5% at 3% haematocrit in a total volume of 200µl of medium RPMI-1640 was determined by Jaswant Singh Bhattacharya (JSB) staining to assess the percent parasitaemia (rings) and uniformly maintained with 50% RBCs (O+).
A stock solution of 5mg/ml of each of the test samples was prepared in DMSO and dilutions were prepared with culture medium. The diluted samples in 20µl volume were added to the test wells so as to obtain nal concentrations (at vefold dilutions) ranging between 0.4µg/mL to 100µg/mL in duplicate well containing parasitized cell preparation. The culture plates were incubated at 37˚C in a candle jar. After 36 to 40h incubation, thin blood smears from each well were prepared and stained with JSB stain. The slides were microscopically observed to record maturation of ring stage parasites into trophozoites and schizonts in presence of different concentrations of the test agents. The test concentration which inhibited the complete maturation into schizonts was recorded as the minimum inhibitory concentrations (MICs). Chloroquine was used as the reference drug[16, 17].

Molecular docking
Molecular docking study was performed by GLIDE v3.8 (Schrodinger, LLC, New York) module for all thiazole derivatives. While (PDB code:5TBO) to predict the anti-malarial activity of newly tested compounds and preprocessed by using ''protein preparation wizard'' in Maestro module v10.3 (Schrodinger, LLC, New York). Some other steps like generate states and re nement have also been processed for addition of hydrogen atoms and disul de bonds at the missing sites on protein molecule. The protein structure was changed to a single unit with the removal of water molecules and other unwanted subunits. After the optimization process, receptor grid generation was proceeded to the previously attached ligand site. The grid is associated with different sets of elds for determining shape and properties of the receptor. The ligand binds with protein residues by using speci c force eld, generate different poses. The best docked poses have been ranked on the basis of energy function combining empirical and force-eld terms [23][24][25][26].

Chemistry
In the present study, 10 thiosemicarbazone and thiazole derivatives have been synthesized which are outlined in scheme 1 and scheme 2. The starting material thiosemicarbazone (1) was prepared by the condensation reaction of substituted aldehyde/ketones with substituted thiosemicarbazides in the presence of ethanol/methanol with few drops of glacial acetic acid reluxing with stirring at 80-90ºC for 8-9 hrs. The thiazole derivatives (2) was prepared by the reaction between thiosemicarbazone derivatives and phenacyl bromide re uxing in isopropyl alcohol for 5 hrs. General procedure for the synthesis of thiosemicarbazone and thiazole derivatives (Kp1-10) were achieved through the versatile and e cient synthetic route outlined in scheme 1 and scheme 2. Brie y, the synthesis of thiosemicarbazone and thiazole was achieved by using substituted aldehyde / ketone, in ethanol / methanol. Whereas, substituted thiosemicarbazides were added with stirring to ethanolic warm solution of substituted aldehyde / ketone in presence of glacial acetic acid. The resulting mixture was re uxed with stirring at 80-90°C and in second step thiosemicarbazones was further treated with phenacyl bromide and the purity of the product as well as composition of reaction mixture were monitored by TLC (Thin layer chromatography). The reaction mixture were cooled down to room temperature and then recrystallized with suitable solvent. The purity of the synthesized thiazoles was ascertained by TLC using Pre-coated Merk silica plate and Ethyl Acetate : Hexane (3 : 2) or chloroform : Hexane (3: 2) as the mobile phase. The spots were detected under UV at 245nm and R f value was calculated. The structures assigned to the compounds were supported by the results of IR, 1 H NMR, 13 C NMR and mass spectral data.
Structure of all the synthesized compounds was monitored by the FTIR, 1 HNMR, 13

In vitro antimalarial activity screening
The antimalarial activity of the synthesized thiazole derivatives (Kp 1-10) was assessed against human pathogenic malarial strain viz. Plasmodium falciparum while quinine was taken as the standard drug.
Activity pro le of all the compounds screened for antimalarial activity is represented in Table 1. Results antimalarial evaluation revealed that most of the tested compounds exhibited remarkable inhibitory activity against the tested plasmodium strain. Among them, compound Kp-9 was found to be most promising which exhibited strongest inhibitory activity against P. falciparum with an IC 50 value of 0.29µg/mL which was higher than the reference drug quinine (1.26µg/mL). Compound 10 also presented excellent inhibition of P. falciparum with IC 50 values of 0.59 respectively. Other tested derivatives such as Kp-1, Kp-3 and Kp-4 also exhibited signi cant antimalarial activity with IC 50 values in the range of 0.65, 0.90, 0.85µg/mL. Compound Kp-2 was the least potent candidate of the series with IC 50 value of 5.52 µg/mL.
The structure-activity relationship of the various titled compounds (Kp-1-10) screened for antimalarial activity has been analysed using quinine as the reference drug. Results of in vitro evaluation data indicated that inhibitory activity of the various tested compounds against P. falciparum depends upon the nature and type of substituents introduced at the 5th position of the thiazole core. Substitution with methyl group or bulky groups in compound Kp-2, Kp-6 at R decrease the anti-malarial activity whereas substitution with hydrogen group at same position responsible for increase in activity as shown by compound Kp-2, Kp-6 and Kp-7. Substitution with electron withdrawing group at R 1 increases anti-Page 10/23 malarial activity as shown in compound Kp-9. Substitution at R 2 with hydrophobic group responsible for decrease in activity as shown in compound Kp-5, Kp-7 and Kp-8. Compounds having hydrogen or hydroxyl group at R and R 2 position shown in compound Kp-1 showed promising anti-malarial activity.
The present study highlighted that most of the synthesized thiazole derivatives possessed strong inhibitory potential against human pathogenic malarial strain P. falciparum.

Computational studies
3.3.1. Pharmacokinetic (ADME) properties prediction The ADME properties of all previously synthesized compounds have been calculated in comparison with active molecules ( Table 2). It has been observed that though all the ADME properties of compounds are well within the acceptable range. Here, proposed compounds showed high percentages of human oral absorption and partition coe cient. Furthermore, synthesized derivatives also showed good cell permeability. From all these results it has been observed that these analogs by making no changes in the core fused scaffold and isosteric/ bioisosteric and knowledge based side chains and fragment attachment came up with very potential lead molecules with favorable drug-like pro le which can emerge as a potential drug molecule in further development. Qikprop predictions suggested that thiazole derivatives have optimum parameters for anti-malarial activity and can be considered as a lead molecule for further modi cations.  10) were docked for studying the essential interactions of compounds with protein to produce antimalarial activity. All the thiazole derivatives were docked (PDB 5TBO) for studying the binding mode of compounds for anti-malarial activity. The potent thiazole derivatives Kp-9, showed highest docking score such as (-9.519) Therefore, compounds Kp-1, Kp-3, Kp-4 and Kp-10 also showed highest docking Score(-8.764, -8.406, -9.062, -9.435 respectively) and critical interactions with VAL532, ILE237, LEU531, HIE185, TYR528, ASN274, ARG265 whereas yellow colour shows H-bond interactions, Purple colour shows Halogen bond, green colour shows pi-cation, green colous shows hydrophobic interactions, blue colour shows polar, green dotted lines shows Pi-Pi stacking.

Conclusion
In the present study, a series of thiosemicarbazone and thiazole derivatives have been synthesized, characterized, and screened against potential anti-malarial activity. The anti-malarial activity of the synthesized thiazole derivatives (Kp1-10) was assessed against human pathogenic malarial strain viz.
Plasmodium falciparum while quinine was taken as the standard drug. Compound Kp-9 was found to be most promising which exhibited strongest inhibitory activity against P.falciparum with an IC 50 value of 0.29µg/mL which was higher than the reference drug quinine (1.26µg/mL). The SAR study revealed that the substitution with electron withdrawing group at phenyl increases anti-malarial activity as shown in compound Kp-9. The result of molecular docking studies showed that compounds Kp-9, Kp-1, Kp-3, Kp-4 has crystal alignment as crystal ligand of protein(PDB code: 5TBO) and compound Kp-9 showed highest docking score such as (-9.519) Therefore, compounds Kp-1, Kp-3, Kp-4 and 10also showed highest docking Score(-8.764, -8.406, -9.062, -9.435 respectively) and critical interactions with VAL532, ILE237, LEU531, HIE185, TYR528, ASN274, ARG265. The results of biological activity and docking study revealed that the presence of electron withdrawing group at 4th position of phenyl ring attached is crucial for better anti-malarial activity and favorable drug-like pro le which can emerge as a potential drug molecule in further development. In conclusion, the structural features of compound Kp-9 may be considered for the development of newer anti-malarial agents.

Declarations
Con ict of Interest: The authors declare no con ict of interest.